MicroRNA-16 sensitizes breast cancer cells to paclitaxel through suppression of IKBKB expression

Abstract

Paclitaxel (Taxol) is an effective chemotherapeutic agent for treating breast cancer patients. However, chemoresistance is a major obstacle in cancer treatment. Here, we showed that overexpression of miR-16 promoted Taxol-induced cytotoxicity and apoptosis in breast cancer cells. Furthermore, IκB kinase β (IKBKB) was identified as a direct target of miR-16. Up-regulation of IKBKB suppressed Taxol-induced apoptosis and led to an increased resistance to Taxol, and restoring IKBKB expression in miR-16-overexpressing breast cancer cells recovered Taxol resistance. Moreover, miR-16 was highly expressed in Taxol-sensitive breast cancer tissues compared with Taxol-resistant tissues, and there was an inverse correlation between miR-16 expression and IKBKB expression in breast cancer tissues. The expression levels of miR-16 were negatively associated with T stages, whereas the expression of IKBKB was positively correlated with T stages, lymph node metastasis and clinical stages. Taken together, our data demonstrates that miR-16 sensitizes breast cancer cells to Taxol through the suppression of IKBKB expression, and targeting miR-16/IKBKB axis will be a promising strategy for overcoming Taxol resistance in breast cancer.

Keywords: IKBKB; Taxol; breast cancer; chemosensitivity; miR-16.

Figure 1. Involvement of miR-16 in Taxol chemosensitivity in breast cancer cells

A. miR-16 levels were detected in MDA-MB-231 and MCF-7 cells transfected with 50 nM miR-16 mimics or 50 nM miR-NC by qRT-PCR. B. MDA-MB-231 and MCF-7 cells transfected with 50 nM miR-16 mimics or 50 nM miR-NC were seeded into 96-well plates and treated with 0, 20, 40 nM (MDA-MB-231) or 0, 200, 400 nM (MCF-7) Taxol for 48 h. The cell viabilities were detected by MTT assays. C. MDA-MB-231 and MCF-7 cells transfected with 50 nM miR-16 mimics or 50 nM miR-NC were seeded into 96-well plates and treated with 0, 10, 20, 40, 80 nM (MDA-MB-231) or 0, 100, 200, 400, 800 nM (MCF-7) Taxol for 48 h. The cell viabilities were detected by MTT assays. D. MDA-MB-231 and MCF-7 cells transfected with 0, 10, 25, 50, 100 nM miR-16 mimics or equal dose of miR-NC were seeded into 96-well plates and treated with 0, 40 nM (MDA-MB-231) or 0, 400 nM (MCF-7) Taxol for 48 h. The cell viabilities were detected by MTT assays. E. MDA-MB-231 and MCF-7 cells transfected with 100 nM miR-16 inhibitor or 100 nM anti-miR-NC (top) were seeded into 96-well plates and treated with 0, 10, 20, 40, 80 nM (MDA-MB-231) or 0, 100, 200, 400, 800 nM (MCF-7) Taxol for 48 h. The cell viabilities were detected using MTT assays. Data are presented as the percentage of viability inhibition measured in untreated cells. Columns, means of three independent experiments; bars, SE. *, p<0.05, **, p<0.01, ***, p<0.001.

Figure 2. Involvement of miR-16 in Taxol-induced apoptosis in breast cancer cells

A. MDA-MB-231 and MCF-7 cells transfected with 50 nM miR-16 mimics or 50 nM miR-NC were treated with 0, 20, 40 nM (MDA-MB-231) or 0, 200, 400 nM (MCF-7) Taxol for 48 h. The cellular morphologies were visualized using a phase-contrast microscope. B-C. MDA-MB-231 and MCF-7 cells were transfected with 50 nM miR-NC or miR-16 mimics and then treated with 40 and 400 nM Taxol for 48 h, respectively. Cell lysates were extracted for western blotting using an antibody against c-PARP (B), or cells were collected for annexin V staining and flow cytometry assays (C). The gray density was quantified using the ImageJ software and normalized to β-actin. The percentage of apoptotic cells is represented in a bar diagram from three independent experiments. D. MDA-MB-231 and MCF-7 cells transfected with 100 nM anti-miR-NC or miR-16 inhibitor were treated with 40 and 400 nM Taxol for 48 h, respectively. Cell lysates were extracted for western blotting using an antibody against c-PARP. β-actin was used as an internal control. Columns, means of three independent experiments; bars, S.E. *, p<0.05, **, p<0.01.

Figure 3. IKBKB is a direct target of miR-16 in breast cancer cells

A. Schematic description of interaction between IKBKB and miR-16. B. MDA-MB-231 and MCF-7 were transfected with 50 nM miR-NC or miR-16 mimics. Cell lysates were prepared for western blotting with an antibody against IKBKB 48 h after transfection. β-actin was used as a loading control. The gray density was quantified using the ImageJ software and normalized to β-actin. C. 0, 25, 50, 100 nM miR-16 mimics were transfected into MDA-MB-231 and MCF-7 cells, and then the expression of IKBKB was detected by western blotting assay as (B) performed. D. MDA-MB-231 and MCF7 were transfected with 100 nM anti-miR-NC or miR-16 inhibitor, and then similar experiments as in (B) were performed. E. MDA-MB-231 and MCF-7 were transfected with 50 nM miR-NC or miR-16 mimics. Total cellular RNA was collected and IKBKB mRNA levels were measured by qRT-PCR 24 h after transfection. The relative mRNA levels of IKBKB were shown in the bar diagram from three independent experiments and normalized to GAPDH. F. MDA-MB-231 and MCF-7 were transfected with 100 nM anti-miR-NC or miR-16 inhibitor, and similar experiments as in (E) were performed and analyzed. G. MDA-MB-231 and MCF-7 were co-transfected with pMIR-IKBKB-3′UTR-wt or pMIR-IKBKB-3′UTR-mut and 50 nM miR-16 mimics using Lipofectamine 3000 reagent. Luciferase activity was measured 48 h after transfection. The pRL-TK vector was used as an internal control. The results were expressed as relative luciferase activity (firefly luc/renilla luc). H. MDA-MB-231 and MCF-7 cells were co-transfected with pMIR-IKBKB-3′UTR-wt or pMIR-IKBKB-3′UTR-mut and 100 nM miR-16 inhibitor using Lipofectamine 3000 reagent, and similar experiments as in (G) were performed and analyzed. Columns, means of three independent experiments; bars, S.E. *, p<0.05, **, p<0.01.

Figure 4. IKBKB plays a critical role in Taxol-induced apoptosis

A. MDA-MB-231 and MCF-7 cells transfected with blank-plasmid or IKBKB (inset) overexpression plasmid were seeded into 96-well plates and treated with 0, 10, 20, 40, 80 nM (MDA-MB-231) or 0, 100, 200, 400, 800 nM (MCF-7) Taxol for 48 h. The inhibition of cell viabilities was detected using MTT assays. Columns, means of three independent experiments; bars, S.E. *p<0.05. B-C. MDA-MB-231 and MCF-7 cells were transfected with blank-plasmid or IKBKB overexpression plasmid and then treated with 40 and 400 nM Taxol for 48 h, respectively. Cell lysates were extracted for western blotting using antibodies against c-PARP and IKBKB (B), or cells were collected for annexin V staining and flow cytometry assays (C). The percentage of apoptotic cells is represented in a bar diagram from three independent experiments (C, right). β-actin was used as a loading control. Columns, means of three independent experiments; bars, S.E. *, p<0.05, **, p<0.01.

Figure 5. Restoring the expression of IKBKB recovers Taxol resistance and counteracts miR-16-mediated Taxol sensitivity

A. MDA-MB-231 and MCF-7 cells were transfected with miR-16 mimics alone or miR-16 mimics plus IKBKB overexpression plasmid and then treated with 40 and 400 nM Taxol for 48 h, respectively. Cell lysates were extracted for western blotting using antibodies against c-PARP and IKBKB. β-actin was used as a loading control. B. MDA-MB-231 and MCF-7 cells transfected with miR-16 mimics alone or miR-16 mimics plus IKBKB overexpression plasmid were seeded into 96-well plates and treated with 0, 20, 40 nM (MDA-MB-231) or 0, 200, 400 nM (MCF-7) Taxol for 48 h. The cell viabilities were then detected using MTT assays. Columns, means of three independent experiments; bars, S.E. *, p<0.05. C. MDA-MB-231 and MCF-7 cells were transfected with miR-16 mimics alone or miR-16 mimics plus IKBKB overexpression plasmid and then treated with 40 and 400 nM Taxol for 48 h, respectively. Cells were collected for annexin V staining and flow cytometry assays. Percentages of apoptotic cells are represented in bar diagram from three independent experiments. Columns, means of three independent experiments; bars, S.E. *, p<0.05.

Figure 6. The expression level of miR-16 in Taxol-sensitive breast cancer tissues is higher than that in Taxol-resistant tissues

A. miR-16 expression was measured by in situ hybridization in paraffin-embedded Taxol-treated breast cancer tissues. Representative cases of Taxol-sensitive (n=27) and Taxol-resistant (n=15) patients with high or low miR-16 staining are shown. B. Proportion of Taxol-treated breast cancer patients with low or high miR-16 expression. *, p<0.05.

Figure 7. MiR-16 expression is inversely correlated with IKBKB expression and their expression levels are associated with clinicopathological characteristics of breast cancer patients

A. Representative images of inverse correlation between miR-16 and IKBKB expression are shown. B. Spearman rank test of 90 breast cancer patients was used for depicting the correlation between miR-16 and IKBKB. C-D. Proportion of breast cancer patients with miR-16 expression and T stages (C) or IKBKB expression and clinical stages (D). E-F. Representative images of breast cancer patients with miR-16 expression and T stages (E) or IKBKB expression and clinical stages (F).